The Clostridium perfringens epsilon toxin, a Category B Select Agent, is responsible for a severe, often fatal enterotoxemia characterized by cardiac, pulmonary, kidney, and brain edema. The mechanism by which epsilon toxin acts is incompletely understood. However, it is believed that toxin monomers bind to specific receptors on sensitive cells, assemble into oligomeric complexes, and form pores in the cell membrane. We hypothesize that inhibitors can be prepared that block one or more of these key steps in the process by which the C. perfringens epsilon toxin mediates cytotoxic effects. The specific aims of this proposal are designed to develop inhibitors of epsilon toxin activity by (1) identifying and analyzing dominant-negative mutant proteins, (2) identifying small molecule inhibitors of epsilon toxin activity, and (3) identifying host cell factors required for epsilon toxin activity. In aim 1, amino acid deletions, insertions, and substitutions will be introduced into the gene encoding epsilon toxin, with special emphasis placed on a region of the protein believed to insert into the target cell membrane. Recombinant mutant proteins will be examined for both a lack of cytotoxicity and for the ability to inhibit the cytotoxic activity of wild- type epsilon toxin. Mutants identified in this aim will increase our understanding of the structure-function relationships that govern epsilon toxin activity, will provide insight into the nature of dominant-negative mutant toxins, and identify new therapeutic candidates for countering exposure to epsilon toxin. In aim 2, a high- throughput screen will be used to identify small molecules that inhibit the cytotoxic activity of epsilon toxin. In addition to identifying novel inhibitors, this aim will validate high-throughput assays that may be used in future studies to test additional compounds for the ability to either inhibit toxin activity or mitigate the effects of the toxin. In aim 3, proteomic analyses of epsilon toxin-sensitive and resistant MDCK cells lines will be used to identify host cell factors required for epsilon toxin activity. This approach is expected to identify the cell-surface receptor recognized by the toxin as well as other host factors that may be targeted for therapeutic intervention. The inhibitors identified in this proposal will provide the foundation for future studies aimed at optimizing the inhibitory activities and testing the effectiveness of the inhibitors using established animal models. PUBLIC HEALTH RELEVANCE The Clostridium perfringens epsilon toxin is one of the most potent bacterial toxins. The U.S. Department of Health and Human Services and the Department of Agriculture have classified the epsilon toxin as a select agent. Currently, there is no countermeasure approved for use in humans and the specific aims of this proposal are designed to develop inhibitors of epsilon toxin activity.